Microscopic pathology defines many human diseases, but the chemical composition of most histologically observed subcellular structures remains largely uncharacterized. Currently, there is no method for isolating subcellular areas of interest without disrupting the cells and thus distorting naturally occurring protein interactions. We propose to combine in situ labeling schemes and subsequent microdissection with mass spectrometry to directly analyze subcellular complexes and organelles. To this end, we are improving expression microdissection (xMD) in order to isolate optically dark targets (chemical, antibody, or metal-based stains) using optimized protocols compatible with mass spectrometric analysis to allow unsupervised thermoplastic capture of subcellular structures. We conducted two types of experiments for initial goals (1) an unsupervised harvest of the apical surface of polarized epithelial cells (MDCK cells);(2) immunohistochemical (IHC) staining with antibodies to CD31, cytokeratin and NeuN on mouse/rat liver, kidney and brain tissues, followed by Laser Capture Microdissection (LCM). At the same time, protein isolation and mass spectrometric methods are being optimized to identify the products from both isolation techniques. Toward the first objective, we produced and tested different types of ethylene-vinyl acetate (EVA) film in order to better control dimensions of material capture. We used spin-coating from toluene solutions with varied percentages (4 to 10% w/v) of EVA to generate films ranging from 0.5 to 2.5 micron supported on rigid backing polymers (25 micron thick absorbing polyimide tapes) and determined capture of the apical surface of MDCK cells. Compared with Elvax 410 (Dupont), the ethylene-vinyl acetate copolymer commercially used for LCM, Elvax 4310 is an ethylene-vinyl acetate/acid terpolymer with a similar melt index and higher solubility; the enhanced solubility makes it easier to use to generate well-controlled films of varying thicknesses by spin-coating. The results showed that a larger amount and greater uniformity of MDCK cells are transferred using the Elvax 4310. Varying the thickness of EVA4310 (from 1.7 to less than 1 micron) was tested to achieve the optimal specificity of transferred apical surface without inclusion of cell nuclei (z-axis of capture). We achieved some specific transfer of the apical membrane with thinner EVA polymers (4-5%). The method of cold-induced microtubule depolymerization was employed in efforts to reduce the mechanical cohesiveness of the cell monolayer and thus aid in the removal of apical surface with fewer nuclei captured. We needed to determine whether contamination from exogenous antibodies added during IHC staining and the chemical alteration of diaminobenzidene (DAB) would preclude subsequent mass spectrometric analyses. To address these concerns, experiments were designed to evaluate the effect of IHC on protein recovery. Ten or 20 micron thick mouse or rat tissues were IHC-stained targeting blood vessels, cytoplasm and nuclei. Stained tissues were dissected with the area of 8 million sq. micron(<3 microgram protein) using an Arcturus Pix Cell II and captured onto EVA membranes with 20 micron thickness. The samples were digested with trypsin using an optimized on-film digestion protocol and subsequently analyzed by nano-HPLC-electrospray ionization-tandem mass spectrometry. The acquired mass spectra were searched to assess the effects of contamination as well as species specific peptides. Compared with hematoxylin and eosin stained or unstained tissues, there is no significant change in the number of identified peptides or proteins for immuno-stained tissue. The taxonomy non-specified searches show that 1% of identified peptides are from environmental contaminants, such as keratin and less than 2% are from exogenous antibodies or other staining reagents. Comparisons between immuno-stained tissue and unstained tissue show that 75% of identified proteins overlapped both protein lists. The observation of LCM dissected immuno-stained tissue gives us the confidence that IHC staining has little negative impact on protein recovery using mass spectrometry-based proteomic analysis. In contrast, artifact contaminants and a high chemical background frequently limit successful complex characterization using immunoprecipitation. In future work, we will test whether in situ enzymes provide improved specificity for staining and capture of the apical surface. Clonal MDCK cell lines stably expressing GPI-HRP target the anchored enzyme to the apical plasma membrane;subsequent treatment with DAB substrate results in a brown precipitate on the membrane. Preliminary tests with this method show that better transfer specificity is observed compared to other methods. The second focus is on microdissection of neuronal nuclei of rat cerebellum using an xMD prototype instrument. Preliminary microdissection results indicate a good specificity for capture of nuclei. Techniques for tissue slicing and IHC staining are being optimized to obtain consistent capture of nuclei for mass spectrometric proteomic analysis.